The major objective of this proposal is the biochemical identification and characterization of the primary defect in a group of mutants which are pleiotropic for the utilization of alternate carbon sources in place of glucose. These pleiotropic mutants isolated from Chinese hamster lung cells in culture have been genetically well characterized. They appear to be caused by a mutation at a single locus and have been grouped into nine complementation units. Preliminary studies suggest that the pleiotropic carbohydrate mutants are defective in oxidative energy production due to a mutation affecting a citric acid cycle enzyme, component of the electron transport chain, or oxidative phosphorylation. A systematic procedure for the identification of the primary lesion is outlined using wild-type cells, revertants, and complementing cell hybrids as controls for mutant cells. Temperature-sensitive mutants grown at permissive and non-permissive temperatures will be very useful in these studies. Biochemical characterization of the properties of the modified protein in the mutants will distinguish between a mutation in a regulatory gene controlling transcription or translation and a mutation in a gene affecting structure and/or activity. In depth biochemical analysis of the modified protein may provide new information about the structural and functional relationships of the affected protein. The rate of glycolysis is expected to be elevated in the mutants compared to the wild-type cells and factors controlling this increased rate will be examined. Since cells in culture, like tumor cells, have a high rate of glycolysis, a study of these regulatory factors might have implications for the rate of tumor growth.